Androflor REAL-TIME PCR Detection Kit. Androflor Screen REAL-TIME PCR Detection Kit USER MANUAL

Transcription

1 For professional use only Androflor REAL-TIME PCR Detection Kit Androflor Screen REAL-TIME PCR Detection Kit USER MANUAL "DNA-Technology, Research & Production" LLC Russia, , Moscow Region, Protvino, 20 Zheleznodorozhnaya Street, Phone/fax:+7(495) (4967) , Customer service department: +7(800) R1-P809-S3/6EU R1-P810-S3/5EU

2 Table of contents 1. INTENDED USE 3 2. METHOD 3 3. CONTENT 3 4. REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 6 5. WARNINGS AND PRECAUTIONS 7 6. SAMPLES 8 7. CONTROLS 8 8. PROCEDURE 9 9. DATA ANALYSIS TROUBLESHOOTING STORAGE AND HANDLING REQUIREMENTS SPECIFICATIONS QUALITY CONTROL KEY TO SYMBOLS 14 2

3 1. INTENDED USE The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are intended for research and diagnostic applications. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are in vitro Nucleic Acid Test (NAT) based microorganisms detection products. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are designed to detect the total bacterial DNA (total bacterial mass), DNA of the opportunistic and true pathogens in men s urogenital tract by multiplex Real-Time Polymerase Chain Reaction (PCR) method. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are used for the study of men s urogenital tract microbiocenosis, results obtained with the kits can be used for the diagnosis of male urogenital tract inflammatory diseases. Depending on the number of identified microorganisms kit is available in the following formats: - Androflor REAL-TIME PCR Detection Kit - Androflor Screen REAL-TIME PCR Detection Kit. Androflor Screen is a shortform of Androflor. 2. METHOD The implemented PCR method is based on amplification of a target DNA sequence. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are based on fluorescent modification of the PCR method. The PCR-mix contains target-specific probes bearing reporter and quencher molecules. Once hybridized to a target sequence, the probe becomes activated. As a result of activation fluorescence increases proportionally to target sequence amplification. The intensity of fluorescence is measured at every cycle of reaction with a Real-time PCR thermal cycler data collection unit and analyzed with the software provided. Defined tubes contain ROX dye label Marker. It tags the tube/strip orientation. The automatic analysis available on DNA-Technology made instruments and software. Analysis is based on the ratio between quantities of different microorganisms groups. 3. CONTENT 3.1 Number of tests The Androflor REAL-TIME PCR Detection Kit is used to perform 12 tests, including positive and negative controls. The Androflor Screen REAL-TIME PCR Detection Kits is used to perform 24 tests, including positive and negative controls. 3

4 3.2 Kit contents Table 1. Kit components Format Androflor Androflor Screen Reagent Number of tubes Amount per tube Number of tubes Amount per tube Paraffin sealed PCR-mix Strip tubes strips 20 μl Strip tubes strips 20 μl 24 8-tubes strips 20 μl Taq-polymerase solution 4 tubes 500 μl 4 tubes 500 μl Mineral oil 4 tubes 1,0 ml 4 tubes 1,0 ml Positive control C+ Expendables: Caps for stripped tubes 1 tube 160 μl 1 tube 160 μl 24 8-cap strips 4

5 Table 2. Androflor REAL-TIME PCR Detection Kit strips content, fluorescent chemistry/detection channels, color tags of tube Detection channel Fam Hex Rox Cy5 Color of the buffer/ paraffin Strip 1 1 Total bacterial mass (TBM) IC Marker - 2 Lactobacillus spp. IC Gardnerella vaginalis - 3 Staphylococcus spp. IC Streptococcus spp. - 4 Megasphaera spp. / Veillonella spp. / Dialister spp. IC Sneathia spp. / Leptotrichia spp. / Fusobacterium spp. IC Ureaplasma urealyticum IC 7 Mycoplasma hominis IC 8 Bacteroides spp. / Porphyromonas spp. / Prevotella spp. IC Ureaplasma parvum Mycoplasma genitalium Atopobium cluster 1 Corynebacteriu m spp. Strip 2 1 Anaerococcus spp. IC Peptostreptococcus spp. / Parvimonas spp. IC Marker - 3 Eubacterium spp. IC Haemophilus spp. IC - - Pseudomonas 5 aeruginosa/ralstonia spp./ IC - - Burkholderia spp. 6 Enterobacteriaceae spp. / Enterococcus spp. 7 Candida spp. IC - 8 Trichomonas vaginalis IC IC - - Neisseria gonorrhoeae - - Human genomic DNA Chlamydia trachomatis Blue/White Colorless/ White Blue/Blue Colorless/ Blue 1 - Atopobium cluster includes: Atopobium spp., Olsenella spp., Collinsella spp. 5

6 Table 3. Androflor Screen REAL-TIME PCR Detection Kit strips content, fluorescent chemistry/detection channels, color tags Detection channel of tube Fam Hex Rox Cy5 Color of the buffer/ paraffin 1 Total bacterial mass (TBM) IC Marker - 2 Lactobacillus spp. IC Gardnerella vaginalis - Blue/White 3 Staphylococcus spp. IC Streptococcus spp. - 4 Ureaplasma urealyticum IC Ureaplasma parvum Corynebacterium spp. 5 Mycoplasma hominis IC 6 Enterobacteriaceae spp./ Enterococcus spp. 7 Candida spp. IC IC Mycoplasma genitalium Human genomic DNA - Colorless / White 8 Trichomonas vaginalis IC Neisseria gonorrhoeae Chlamydia trachomatis 4. REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 4.1 Specimen collection Specimen collection swabs: use only dacron, rayon, or calcium alginate tipped collection swabs with plastic or non-aluminum wire shafts. Use DNA-Technology made transport media ( PBS for the transportation of the sample. P-001/1EU) or equivalent or sterile saline or sterile UV PCR cabinet 4.2 DNA extraction and PCR Vortex mixer; 6

7 Household refrigerator with a freezer chamber 0.2, 0.5 and 1.5 ml tubes; PCR tube rack for 0.2, 0.5 and 1.5 ml tubes; Single channel pipettes (volume range μl, μl, μl, μl); RNase and DNase free filtered pipette tips (volume range 20 μl, 50 μl, 200 μl, 1000 μl); Powder-free surgical gloves; Disinfectant solution; Container for used pipette tips; Nucleic acid extraction kit ( DNA-Technology made PREP-NA-PLUS P-002/2EU or PREP-GS-PLUS P-003/2EU are recommended); High-speed centrifuge (13000 rpm); Thermostat (temperature range 50 C -65 C); Real-time PCR thermal cycler ( DNA-Technology made DTprime О-DTPRIME4M1-EU, О- DTPRIME5M1-EU, DTlite ; «Androflor.ini» file). О-DTLITE4S1-EU, О-DTLITE5S1-EU, software version is not lower than 5. WARNINGS AND PRECAUTIONS Handle and dispose all biological samples, reagents and materials used to carry out the assay as if they were able to transmit infective agents. Avoid direct contact with the biological samples reagents and materials used to carry out the assay. Avoid producing spills or aerosol. Any material coming in contact with the biological samples must be treated for at least 30 minutes with disinfecting solution or autoclaved for 1 hour at 121 C before disposal. Molecular biology procedures, such as nucleic acids extraction, reverse transcription, amplification and detection require qualified staff to avoid the risk of erroneous results, especially due to the degradation of nucleic acids contained in the samples or sample contamination by amplification products. All oligonucleotide components are produced by artificial synthesis technology according to internal quality control protocol and do not contain blood or products of blood processing. Positive control is produced by artificial DNA synthesis technology. Positive control does not include parts of infectious agents. All the liquid solutions are designed for single use and can not be used more than once in amplification reactions. Plastic tubes do not contain phthalates. Do not breathe gas/fumes/vapour/spray produced by the components of the kit. Do not eat/drink components of the kit. Avoid contact with eyes. Do not use the kit after the expiry date provided. Only use the reagents provided in the kit and those recommended 7

8 by manufacturer. Do not mix reagents from different batches. Do not use reagents from third party manufacturers kits. Significant health effects are NOT anticipated from routine use of this kit when adhering to the instructions listed in the current manual. 6. SAMPLES The Androflor and Androflor Screen REAL-TIME PCR Detection Kits are designed to detect DNA extracted from the epithelial scrapes from the balanus, urethra; urina; prostatic fluid; ejaculate; biopsy samples from prostatic tissues. The detailed description of sampling and sample processing procedures as well as sample storage and transportation requirements cited in PREP-NA-PLUS and PREP-GS-PLUS Extraction Kits user manuals. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits compatibility with other DNA extraction kits must be confirmed by DNA-Technology company's representative. Regardless of the used DNA extraction kit perform each step of sample preparation procedure for negative control ("C-") in parallel with test samples. Table CONTROLS Control The controlled step Result Interpretation Specific signal + Specific signal - C+ PCR + + Valid - - Invalid 2 C- PCR and DNA extraction + + Valid - - Invalid 2 IC PCR +/- + Valid - Invalid 2 SIC Sampling quality + (sufficient sample amount is determined automatically) - or less than threshold quantity + (sufficient sample amount is determined automatically) - or less than threshold quantity Valid Invalid see TROUBLESHOOTING for more information. 8

9 The sample contains DNA of certain microorganism if the signal for specific DNA is present. The signal for IC could be absent in samples with high concentration of specific DNA due to competitive priming. The sample does not contain DNA of certain microorganism if the signal for specific DNA is absent and for IC and SIC is present. 8. PROCEDURE 8.1 Mark the required number of strips with paraffin-sealed PCR-mix for each test sample, negative control ( C- ) and positive control ( C+ ). Androflor format - two strips with paraffin-sealed PCR-mix (strip 1 and strip 2) are used for one sample analysis, including C- and C+. Androflor Screen format one strip with paraffin-sealed PCR-mix is used for one sample analysis, including C- and C+. Example. An example of strips marking for testing of 2 samples see in the Table 5. Table 5. Marking of strips for PCR Format Androflor Androflor Screen Sample 1 Sample 2 C+ C- Strip 1 Strip 2 Strip 1 Strip 2 Strip 1 Strip 2 Strip 1 Strip 2 Strip with paraffin-sealed PCR-mix Strip with paraffin-sealed PCR-mix Strip with paraffin-sealed PCR-mix Strip with paraffin-sealed PCR-mix 8.2 Vortex the Taq-polymerase solution thoroughly (3-5 sec), then spin briefly (1-3 sec). Add 10 μl of Taq-polymerase solution into each tube. Avoid paraffin layer break. 8.3 Add one drop (~20 μl) of mineral oil into each tube. Close strips tightly. 8.4 Add 5.0 μl of the DNA sample into each tube of a strip (or strips) assigned to test samples the corresponding tubes. Avoid paraffin layer break. Open the strip, add DNA sample, then close the strip before proceeding to the next DNA sample to prevent contamination. Use filter tips. Do not add DNA into the C-, C+ strips. 8.5 Add 5.0 μl of C- which passed whole DNA extraction procedure and C+ into corresponding strips. Avoid paraffin layer break. 8.6 Spin strips briefly at 1000 rpm for 1-3 sec. 8.7 Set the strips to Real-Time PCR Thermal Cycler. Central position of the strips is recommended. 8.8 Launch the Thermal Cycler software and run PCR according to instructions supplied with device. If 9

10 using DNA-Technology made Real-Time PCR Thermal Cycler, launch the RealTime_PCR application in Device handling mode. Upload Androflor.ini file before the first run. Add test in subsequent runs. Specify the number and identificators of samples. Define position of strips in software interface according to position they were set in thermal unit (see p.8.7). Run PCR. See table 6 to refer the cycling program and table 4 to refer the detection channels. Table 6. The PCR program for DTlite and DTprime Thermal Cyclers. Step Temperature, C Min. Sec. Number of cycles Optical measurement Type of the step Cycle Cycle v Cycle v Cycle 5 10 Holding 9. DATA ANALYSIS 9.1 In case of using DNA-Technology made Real-Time PCR Thermal Cyclers the analysis is performed automatically. The controls should be also considered to exclude false positive and false negative results (see p. 7 of the current manual). 9.2 Upon completion of the run, software compares actual position of the strip (by mean of ROX dye label position) relative to position predefined by the operator. If there is a mismatch, the software gives a warning about discrepancy and suggests rearrangement of the tubes by default. 9.3 The quantitative measure (common logarithm of concentration) and diagram are displayed in the line with taxonomic identifier of microorganism detected. For true pathogen result of qualitative analysis (+ or -) is displayed. 9.4 Sample quality validation is estimated automatically and based on the Total bacterial mass and Human genomic DNA values. 10

11 Table TROUBLESHOOTING Specific signal + Specific signal - Possible cause Solution C+ - - Operation error Repeat whole test PCR inhibition Violation of storage and handling requirements Dispose current batch C- + + Contamination Dispose current batch Perform decontamination procedures IC - PCR inhibition Repeat PCR amplification or repeat DNA extraction and PCR amplification or repeat sampling procedure, DNA extraction and PCR SIC < than threshold quantity < than threshold quantity Insufficient sample amount amplification Repeat sampling procedure If you face to any undescribed issues contact our representative in EC. 11. STORAGE AND HANDLING REQUIREMENTS Expiry date 12 months since from the date of Quality Control Department approval in compliance with all transportation, storage and operation conditions. All components of the Androflor and Androflor Screen REAL-TIME PCR Detection Kits must be stored at temperatures between 2 С and 8 С and out of light over the storage period. Transportation can be held by all types of roofed transport at temperatures between 2 С and 8 С over the transportation. An expired Androflor and Androflor Screen REAL-TIME PCR Detection Kits must not be used. We strongly recommend following the instructions to get robust and reliable results. 11

12 The conformity of the Androflor and Androflor Screen REAL-TIME PCR Detection Kits to the prescribed technical requirements is subject to compliance of storage, carriage and handling conditions recommended by manufacturer. Contact our official representative in EU by quality issues of the Androflor and Androflor Screen REAL- TIME PCR Detection Kits. 12. SPECIFICATIONS a. The Androflor and Androflor Screen REAL-TIME PCR Detection Kits allows to determine quantity of microorganisms in transport medium proportional to total microbial content of corresponding biotope in men s urogenital tract. b. The specificity of the Androflor and Androflor Screen REAL-TIME PCR Detection Kits was evaluated by bioinformatic analysis based on available on-line databases containing comprehensive genetic information on the living organisms to date. The oligonucleotides used in the test and determining its specificity was checked against GenBank database sequences including those specific for cultured and uncultured microorganisms inhabiting urogenital tract. None of the sequences showed similarity level sufficient enough to provide unspecific detection. c. The sensitivity of the Androflor and Androflor Screen REAL-TIME PCR Detection Kits for all target microorganisms except Mycoplasma genitalium, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis 4000 copies/ml; for Mycoplasma genitalium, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis 1000 copies/ml Diagnostic features of the Androflor and Androflor Screen REAL-TIME PCR Detection Kits are summarized in the table 8. The claimed specifications are guaranteed when DNA extraction is performed with DNA- Technology made PREP-NA-PLUS P-002/2EU or PREP-GS-PLUS P-003/2EU Kits. 12

13 Table 8. Diagnostic features Sensitivity (95% confidence interval) Specificity (95% confidence interval) Lactobacillus spp. 97.8( ) 98.0( ) Staphylococcus spp. 97.7( ) 97.5( ) Streptococcus spp. 97.5( ) 97.2( ) Corynebacterium spp. 97.9( ) 96.8( ) Gardnerella vaginalis 97.1( ) 97.4( ) Atopobium cluster (Atopobium spp., Olsenella spp., Collinsella spp.) Megasphaera spp. / Veillonella spp. / Dialister spp. Sneathia spp. / Leptotrichia spp. / Fusobacterium spp. 98.9( ) 98.1( ) 98.6( ) 98.2( ) 100( ) 98.7( ) Ureaplasma urealyticum 91.4( ) 97.2( ) Ureaplasma parvum 95.8( ) 98.2( ) Mycoplasma hominis 93.1( ) 98.9( ) Bacteroides spp. / Porphyromonas spp. / Prevotella spp. 99.1( ) 99.2( ) Anaerococcus spp. 99.3( ) 99.3( ) Peptostreptococcus spp. / Parvimonas spp., 99.2( ) 99.3( ) Eubacterium spp. Pseudomonas aeruginosa / Ralstonia spp. / 90.9( ) 97.6( ) Burkholderia spp. Haemophilus spp. 93.4( ) 97.4( ) Enterobacteriaceae spp. / Enterococcus spp. 97.6( ) 96.6( ) Candida spp. 100( ) 98.8( ) Mycoplasma genitalium 100( ) 99.7( ) Trichomonas vaginalis 100( ) 99.8( ) Neisseria gonorrhoeae 100( ) 99.8( ) Chlamydia trachomatis 100( ) 99.6( ) 13. QUALITY CONTROL DNA-Technology, Research&Production LLC declares that the above mentioned products meet the provision of the Council Directive 98/79/EC for In Vitro Diagnostic Medical Devices. The quality control procedures performed in accordance with ISO ISO 9001:2008 and ISO 13485:

14 14. KEY TO SYMBOLS Authorized representative in EU Upper limit of temperature Caution Manufacturer Consult instructions for use Negative control Date of manufacture Positive control Expiration date Cataloque number In vitro diagnostic medical device Sufficient for Batch code Temperature limitation Version R1-P809-S3/6EU R1-P810-S3/5EU

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